Project Summary/Abstract The ultimate objective of the proposed research is to enable the long-term banking of curative doses of human pancreatic islets for the reversal of type 1 diabetes and the amelioration of type 2 diabetes and to understand storage injury near the glass transition temperature (TG) to enable a new type of long term storage to be developed. Banking should reduce or prevent the deterioration of islets prior to use, facilitate tolerance induction, enable delayed islet transplantation following transplantation of a kidney from the same donor weeks earlier, and have many other benefits. We have already demonstrated, in unpublished results, that it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viability (based on vital staining), glucose-stimulated insulin release (stimulation index), glucose-stimulated oxygen consumption, and ability to reverse induced diabetes in mice with minimal islet doses, and we are working on scaling up from 13,000 IEQs per vitrified batch to full curative doses of islets (450,000-720,000). The next step is to establish that islets remain highly viable and functional following at least 3-4 weeks of storage and to determine the best storage conditions for islets. The best storage conditions are postulated to be quite unconventional due to the large volumes associated with vitrifying all islets from a single donor, the fact that the islets will be vitrified, and the desirability of rapid transfer into and out of long term storage without any risk of fracturing. In particular, we believe that islets should be stored near TG, and possibly even above TG, but storage in this temperature range has not been previously studied in any adequate way. Accordingly, Aim 1 is devoted to determining the effect of temperature on islet viability and functionality, Aim 2 is directed toward determining the effect of temperature on ice nucleation in both the islets and their vitrified medium and the relationship between ice nucleation and islet integrity, and Aim 3 is intended to determine the effect of storage time beyond 1 month. One month of storage should be sufficient for most islet banking needs, but two months would be very valuable for providing a safety margin and more flexibility to the clinician and the patient. At the same time, comparing 1 and 2 months of storage will open up information about rates of nucleation over time near TG that is presently entirely lacking, and will begin to answer questions about biological stability near TG that presently remain entirely obscure.